The DNA polymerase III holoenzyme of E. coli is a prototypical replicative complex, exhibiting properties in common with other cellular replicases, including a high rate of processive elongation and the ability to interact with other proteins at the replication fork, establishing the communication channels necessary to coordinate the events required for efficient chromosomal replication. A key component of all cellular replicases is a multi-subunit assembly of homologous proteins that require ATP to assemble a 'sliding clamp processivity factor' onto primer termini. In E.coli, this function is served by the DnaX complex, DnaX3-delta-delta-chi-psi. The dnaX gene of E.coli encodes two distinct products: tau, the full-length translation product and gamma, a shorter protein that arises by translational frameshifting. In spite of gamma and tau being found together within holoenzyme, they do not readily form mixed complexes in vitro or when overexpressed in vivo. During the last grant period, we have determined the limits of five domains of the tau DnaX protein, the three domains of the homologous delta-prime subunit, and have defined the subunit interactions of each domain. During the next grant period, we will determine the factors required for proper DnaX complex assembly using an in vitro assay we have recently developed. We will refine our understanding of the subunit interface domains down to the amino acid level. Lastly, we have developed a system that permits individually assessing the occupancy of each polymerase site of the dimeric holoenzyme with primer-templates. This system will be used to determine whether the internal DnaX clamp loader can assemble beta2 for both strands of DNA at the replication fork, and to further investigate our asymmetric dimer hypothesis. These studies are expected to significantly improve our understanding of the structure and function of this important and highly conserved enzyme system, to provide insight into how living systems assemble their multisubunit complexes that load processivity factors onto DNA, and to help us understand the functional advantages of such complexes.